The long range goal of this project is to improve cryopreservation (CP) procedures for human sperm, so that more sperm competent to fertilize eggs will survive frozen storage, and the success rate of artificial insemination will be increased. The effect of different CP protocols on the pattern and vigor of sperm movement will be determined by multivariate, computer aided sperm analysis, and the hypothesis that changes in movement patterns are due to altered internal calcium, pH or cAMP rather than an altered flagellar motor will be tested. The ability of sperm immobilized by CP to be reactivated by experimentally altered internal calcium, pH, or cAMP will be determined. The effect of a variety of CP protocols on acrosomal function of motile sperm will be determined by studying acrosomal structure, acrosin content, maintenance of an acidic acrosomal interior, acrosome stability during in vitro incubation, and the response of sperm to a stimulator of the acrosome reaction. The ability of CP sperm to bind to the zona pellucida and to fuse with the oolemma will be determined. Fourier transform infrared spectroscopy will be used to detect lipid phase transitions in sperm, and the hypothesis that these transitions are a source of damage during CP will be tested by comparing transition temperature (Tm) for sperm that survive CP this the Tm of sperm that do not. Further, agents known to depress Tm in other systems will be tested for their value as sperm cryoprotectants. The hypothesis that phase separations in the plane of the membrane cause redistribution of lipids and proteins will be tested by freeze-fracture. Results of the cell biology studies will be utilized in the context of an empirical testing program which will select a set of new CP methods for further testing with bioassays of sperm function. The choice of bioassays will be based on paired observations of CP sperm exposed to cervical mucus in vitro and in vivo (i.e., recovered after AI). The functional competence of CP sperm will be determined in these assays by their swimming characteristics, their acrosomal integrity, their survival and pattern of movement during incubation under standard in vitro conditions and their ability to undergo the acrosome reaction in response to a biological stimulus. The selected bioassays will be used to identify a superior CP method from among the candidates generated by the empirical testing. Clinical studies will subsequently be carried out in which the superior CP method will be used for AI of study subjects and sperm will be recovered from the subjects for functional testing in vitro.